Microfluidic Electroporation Arrays for Investigating Electroporation-Induced Cellular Rupture Dynamics.

Electroporation is pivotal in bioelectrochemistry for cellular manipulation, with prominent applications in drug delivery and cell membrane studies. A comprehensive understanding of pore generation requires an in-depth analysis of the critical pore size and the corresponding energy barrier at the onset of cell rupture. However, many studies have been limited to basic models such as artificial membranes or theoretical simulations. Challenging this paradigm, our study pioneers using a microfluidic electroporation chip array. This tool subjects live breast cancer cell species to a diverse spectrum of alternating current electric field conditions, driving electroporation-induced cell rupture. We conclusively determined the rupture voltages across varying applied voltage loading rates, enabling an unprecedented characterization of electric cell rupture dynamics encompassing critical pore radius and energy barrier. Further bolstering our investigation, we probed cells subjected to cholesterol depletion via methyl-ß-cyclodextrin and revealed a strong correlation with electroporation. This work not only elucidates the dynamics of electric rupture in live cell membranes but also sets a robust foundation for future explorations into the mechanisms and energetics of live cell electroporation.

Factors related to tumor response rate from TCGA three omics data-variants, expression, methylation.

The Cancer Genome Atlas (TCGA) and its patient-derived multi-omics datasets have been the backbone of cancer research, and with novel approaches, it continues to shed new insight into the disease. In this study, we delved into a method of multi-omics integration of patient datasets and the association of biological pathways related to the disease. First, across thirty-three types of cancer present in TCGA, we merged genomic mutations and drug response datasets and filtered for the viable variant-drug response combinations available in TCGA, containing more than three samples for each drug response label with RNA sequencing (RNA-seq) and genomic methylation data available for each patient. We identified two distinct combinations in TCGA, one being pancreatic adenocarcinoma patients with/without rs121913529 variant in KRAS gene treated with gemcitabine, and the other low-grade glioma with/without rs121913500 variant in IDH1 gene administered with temozolomide. In these two groups, different patterns of gene expression were observed in the pathways often associated with cancer progression, such as mTOR and PDGF between the patients with complete response and progressive disease. Our result will provide yet another example of the relevance of these biological pathways in cancer drug response and a way for multi-omics integration in cancer datasets.

Proteolysis-driven proliferation and rigidification of pepsin-resistant amyloid fibrils.

Proteolysis of amyloids is related to prevention and treatment of amyloidosis. What if the conditions for proteolysis were the same to those for amyloid formation? For example, pepsin, a gastric protease is activated in an acidic environment, which, interestingly, is also a condition that induces the amyloid formation. Here, we investigate the competition reactions between proteolysis and synthesis of amyloid under pepsin-activated conditions. The changes in the quantities and nanomechanical properties of amyloids after pepsin treatment were examined by fluorescence assay, circular dichroism and atomic force microscopy. We found that, in the case of pepsin-resistant amyloid, a secondary reaction can be accelerated, thereby proliferating amyloids. Moreover, after this reaction, the amyloid became 32.4 % thicker and 24.2 % stiffer than the original one. Our results suggest a new insight into the proteolysis-driven proliferation and rigidification of pepsin-resistant amyloids.

On-Chip Single-Cell Bioelectrical Analysis for Identification of Cell Electrical Phenotyping in Response to Sequential Electric Signal Modulation.

In recent years, an interesting biomarker called membrane breakdown voltage has been examined using artificial planar lipid bilayers. Even though they have great potential to identify cell electrical phenotyping for distinguishing similar cell lines or cells under different physiological conditions, the biomarker has not been evaluated in the context of living cell electrical phenotyping. Herein, we present a single-cell analysis platform to continuously measure the electric response in a large number of cells in parallel using electric frequency and voltage variables. Using this platform, we measured the direction of cell displacement and transparent cell image alteration as electric polarization of the cell responds to signal modulation, extracting the dielectrophoretic crossover frequency and membrane breakdown voltage for each cell, and utilizing the measurement results in the same spatiotemporal environment. We developed paired parameters using the dielectrophoretic crossover frequency and membrane breakdown voltage for each cell and evaluated the paired parameter efficiency concerning the identification of two different breast cancer cells and cell drug response. Moreover, we showed that the platform was able to identify cell electrical phenotyping, which was generated by subtle changes in cholesterol depletion-induced cell membrane integrity disruption when the paired parameter was used. Our platform introduced in this paper is extremely useful for facilitating more accurate and efficient evaluation of cell electrical phenotyping in a variety of applications, such as cell biology and drug discovery.

Increased Anti-Allergic Effects of Secretome of Low-Level Light Treated Tonsil-Derived Mesenchymal Stem Cells in Allergic Rhinitis Mouse Model.

BACKGROUND: Low-level light therapy (LLLT) is widely used for the photobiomodulation of cell behavior. Recent studies have shown that LLLT affects the proliferation and migration of various types of mesenchymal stem cells (MSCs). However, there is a lack of studies investigating the effect of LLT on enhancing the immunomodulatory properties of tonsil-derived MSCs (T-MSCs). OBJECTIVE: The aim of this study was to investigate the immunomodulatory effects of conditioned media from T-MSCs (T-MSCs-CM) treated with LLLT in allergic inflammation. METHODS: We isolated T-MSCs from human palatine tonsils and evaluated the ingredients of T-MSCs-CM. The effect of T-MSCs-CM treated with LLLT was evaluated in a mouse model of allergic rhinitis (AR). We randomly divided the mice into four groups (negative control, positive control, T-MSCs-CM alone, and T-MSCs-CM treated with LLLT). To elucidate the therapeutic effect, we assessed rhinitis symptoms, serum immunoglobulin (Ig), the number of inflammatory cells, and cytokine expression. RESULTS: We identified increased expression of immunomodulatory factors, such as HGF, TGF-ß, and PGE, in T-MSCs-CM treated with LLLT, compared to T-MSCs-CM without LLLT. Our animal study demonstrated reduced allergic symptoms and lower expression of total IgE and OVA-specific IgE in the LLLT-treated T-MSCs-CM group compared to the AR group and T-MSCs-CM alone. Moreover, we found that T-MSCs-CM treated with LLLT showed significantly decreased infiltration of eosinophils, neutrophils, and IL-17 cells in the nasal mucosa and reduced IL-4, IL-17, and IFN-γ expression in OVA-incubated splenocytes compared to the AR group. CONCLUSIONS: The present study suggests that T-MSCs-CM treated with LLLT may provide an improved therapeutic effect against nasal allergic inflammation than T-MSCs-CM alone.

Droplet Microfluidics with MALDI-MS Detection: The Effects of Oil Phases in GABA Analysis.

Microfluidic and mass spectrometry (MS) methods are widely used to sample and probe the chemical composition of biological systems to elucidate chemical correlates of their healthy and disease states. Though matrix-assisted laser desorption/ionization-mass spectrometry (MALDI)-MS has been hyphenated to droplet microfluidics for offline analyses, the effects of parameters related to droplet generation, such as the type of oil phase used, have been understudied. To characterize these effects, five different oil phases were tested in droplet microfluidics for producing samples for MALDI-MS analysis. Picoliter to nanoliter aqueous droplets containing 0.1 to 100 mM γ-aminobutyric acid (GABA) and inorganic salts were generated inside a polydimethylsiloxane microfluidic chip and deposited onto a conductive glass slide. Optical microscopy, Raman spectroscopy, and MALDI-mass spectrometry imaging (MSI) of the droplet samples and surrounding areas revealed patterns of solvent and oil evaporation and analyte deposition. Optical microscopy detected the presence of salt crystals in 50-100 µm diameter dried droplets, and Raman and MSI were used to correlate GABA signals to the visible droplet footprints. MALDI-MS analyses revealed that droplets prepared in the presence of octanol oil led to the poorest detectability of GABA, whereas the oil phases containing FC-40 provided the best detectability; GABA signal was localized to the footprint of 65 pL droplets with a limit of detection of 23 amol. The effect of the surfactant perfluorooctanol on analyte detection was also investigated.

Effect of glutaraldehyde-crosslinked cartilage acellular matrix film on anti-adhesion and nerve regeneration in a rat sciatic nerve injury model.

Decellularized extra-cellular matrix (ECM) has been studied as an alternative to anti-adhesive biomaterials and cartilage acellular matrix (CAM) has been shown to inhibit postoperative adhesion in several organs. This study aimed to evaluate the suitability of glutaraldehyde (GA) crosslinked CAM-films as anti-adhesion barriers for peripheral nerve injury. The films were successfully fabricated and showed improved physical properties such as mechanical strength, swelling ratio, and lengthened degradation period while maintaining the microstructure and chemical composition after GA crosslinking. In the in vitro study of CAM-film, the dsDNA content met the recommended limit of decellularization and more than 70% of the major ECM components were preserved after decellularization. The adhesion and proliferation of seeded human umbilical vein endothelial cells and fibroblasts were significantly lower in CAM-film than in control, but similar with Seprafilm. However, the CAM-film extract did not show cytotoxicity. In the in vivo study, the peri-neural fibrosis was thicker, adhesion score higher, and peri-neural collagen fibers more abundant in the control group than in the CAM-film group. The total number of myelinated axons was significantly higher in the CAM-film group than in the control group. The inflammatory marker decreased with time in the CAM-film group compared to that in the control group, whereas the nerve regenerative marker expression was maintained. Moreover, the ankle angles at contracture and toe-off were higher in the CAM film-treated rats than in the control rats. GA-crosslinked CAM films may be used during peripheral nerve surgery to prevent peri-neural adhesion and enhance nerve functional recovery.

Synergistic effect of three-dimensional coculture and photobiomodulation therapy on vascularized liver spheroid formation by stem cells.

Despite studies reporting functional differentiation of liver cells, a three-dimensional, vascularized liver organ has yet to be developed from mesenchymal stem cells. We investigated whether treatment with photobiomodulation (PBM) before three-dimensional liver spheroid transplantation improved the recovery of liver function via stimulation of angiogenesis and hepatocyte differentiation. Liver spheroids composed of hepatic, endothelial, and mesenchymal cells were subjected to PBM therapy. To evaluate the in vivo therapeutic effect of the liver spheroids treated with PBM, phosphate-buffered saline, liver spheroid, and PBM-treated liver spheroid were transplanted into a damaged host liver using conventional chimeric mouse models. To further characterize the maturation of transplanted PBM-liver spheroid compared with the newly generated non-PBM-liver spheroid or human liver tissues, the expression profiles of mature liver signature genes were analyzed. Liver spheroids expressed hepatocyte growth factors, including vascular endothelial growth factor and angiogenic factors. The cells in liver spheroid compensated for the low viability and improved the function of hepatocytes. Here, we demonstrate the formation of vascularized and functional human liver spheroid from human adipose-derived stem cells by transplantation of liver tissue created in vitro. Albumin secretion by PBM-treated liver spheroid was higher on Day 28 compared with liver spheroid-seeded transplant group. PBM-liver spheroids serve as individual vascularization units, promoting the simultaneous development of new microvascular networks at different locations inside the implanted tissue constructs. The vasculature in the liver spheroid transplants became functional by connecting to the host vessels within 48 h. These PBM-liver spheroids may be useful in designing artificial three-dimensional hepatic tissue constructs and in cell therapy with limited numbers of human hepatocytes.

Variable Membrane Dielectric Polarization Characteristic in Individual Live Cells.

Investigation of the dielectric properties of cell membranes plays an important role in understanding the biological activities that sustain cellular life and realize cellular functionalities. Herein, the variable dielectric polarization characteristics of cell membranes are reported. In controlling the dielectric polarization of a cell using dielectrophoresis force spectroscopy, different cellular crossover frequencies were observed by modulating both the direction and sweep rate of the frequency. The crossover frequencies were used for the extraction of the variable capacitance, which is involved in the dielectric polarization across the cell membranes. In addition, this variable phenomenon was investigated by examining cells whose membranes were cholesterol-depleted with methyl-ß-cyclodextrin, which verified a strong correlation between the variable dielectric polarization characteristics and membrane composition changes. This study presented the dielectric polarization properties in live cells' membranes that can be modified by the regulation of external stimuli and provided a powerful platform to explore cellular membrane dielectric polarization.

A novel automatic segmentation and tracking method to measure cellular dielectrophoretic mobility from individual cell trajectories for high throughput assay.

BACKGROUND AND OBJECTIVE: The dielectrophoresis (DEP) technique is increasingly being recognised as a potentially valuable tool for non-contact manipulation of numerous cells as well as for biological single cell analysis with non-invasive characterisation of a cell's electrical properties. Several studies have attempted to track multiple cells to characterise their cellular DEP mobility. However, they encountered difficulties in simultaneously tracking the movement of a large number of individual cells in a bright-field image sequence because of interference from the background electrode pattern. Consequently, this present study aims to develop an automatic system for imaging-based characterisation of cellular DEP mobility, which enables the simultaneous tracking of several hundred of cells inside a microfluidic device. METHODS: The proposed method for segmentation and tracking of cells consists of two main stages: pre-processing and particle centre localisation. In the pre-processing stage, background subtraction and contrast enhancement were performed to distinguish the cell region from the background image. In the particle centre localisation stage, the unmarked cell was automatically detected via graph-cut algorithm-based K-means clustering. RESULTS: Our algorithm enabled segmentation and tracking of numerous Michigan Cancer Foundation-7 (MCF-7) cell trajectories while the DEP force was oscillated between positive and negative. The cell tracking accuracy and cell count capability was at least 90% of the total number of cells with the newly developed algorithm. In addition, the cross-over frequency was measured by analysing the segmented and tracked trajectory data of the cellular movements caused by the positive and negative DEP force. The measured cross-over frequency was compared with previous results. The multi-cellular movements investigation based on the measured cross-over frequency was repeated until the viability of cells was unchanged in the same environment as in a microfluidic device. The results were statistically consistent, indicating that the developed algorithm was reliable for the investigation of DEP cellular mobility. CONCLUSION: This study developed a powerful platform to simultaneously measure the DEP-induced trajectories of numerous cells, and to investigate in a robust, efficient, and accurate manner the DEP properties at both the single cell and cell ensemble level.

Engineered cartilage utilizing fetal cartilage-derived progenitor cells for cartilage repair.

The aim of this study was to develop a fetal cartilage-derived progenitor cell (FCPC) based cartilage gel through self-assembly for cartilage repair surgery, with clinically useful properties including adhesiveness, plasticity, and continued chondrogenic remodeling after transplantation. Characterization of the gels according to in vitro self-assembly period resulted in increased chondrogenic features over time. Adhesion strength of the cartilage gels were significantly higher compared to alginate gel, with the 2-wk group showing a near 20-fold higher strength (1.8 ± 0.15 kPa vs. 0.09 ± 0.01 kPa, p < 0.001). The in vivo remodeling process analysis of the 2 wk cultured gels showed increased cartilage repair characteristics and stiffness over time, with higher integration-failure stress compared to osteochondral autograft controls at 4 weeks (p < 0.01). In the nonhuman primate investigation, cartilage repair scores were significantly better in the gel group compared to defects alone after 24 weeks (p < 0.001). Cell distribution analysis at 24 weeks showed that human cells remained within the transplanted defects only. A self-assembled, FCPC-based cartilage gel showed chondrogenic repair potential as well as adhesive properties, beneficial for cartilage repair.

The Supernatant of Tonsil-Derived Mesenchymal Stem Cell Has Antiallergic Effects in Allergic Rhinitis Mouse Model.

METHODS: We isolated T-MSCs from human palatine tonsil and evaluated the ingredients of T-MSCs-CM. The effect of T-MSCs-CM was evaluated in the AR mouse model that was randomly divided into five groups (negative control, positive control, and T-MSCs-CM treated (0.1 mg, 1 mg, and 10 mg)). To investigate the therapeutic effect, we analyzed rhinitis symptoms, serum immunoglobulin (Ig), inflammatory cells, and cytokine expression. We also assessed T cell receptor signal, including MAP kinase (ERK/JNK), p65, and NFAT1. RESULTS: We identified the increment of TGF-ß1, PGE2, and HGF in the T-MSCs-CM. In an animal study, the T-MSCs-CM-treated group showed significantly reduced allergic symptoms and infiltration of eosinophils and neutrophils in the nasal mucosa, whereas there was no significant difference in total IgE and the OVA-specific IgE level. Additionally, we found that the 10 mg T-MSCs-CM-treated group showed a significantly decreased IL-4 mRNA expression, compared to the (+) Con group. In the analysis of T cell receptor signal, the phosphorylation of MAP kinases, translocation of p65, and activation of NFAT1 were inhibited after T-MSCs-CM. CONCLUSIONS: Our findings suggest that T-MSCs-CM showed a partial immunomodulatory effect on the AR mouse model by the inhibition of T cell activation via MAP kinase, p65, and NFAT1.

In Vivo Bioreactor Using Cellulose Membrane Benefit Engineering Cartilage by Improving the Chondrogenesis and Modulating the Immune Response.

BACKGROUND: To regenerate tissue-engineered cartilage as a source of material for the restoration of cartilage defects, we used a human fetal cartilage progenitor cell pellet to improve chondrogenesis and modulation of the immune response in an in vivo bioreactor (IVB) system. METHODS: IVB was buried subcutaneously in the host and then implanted into a cartilage defect. The IVB was composed of a silicone tube and a cellulose nano pore-sized membrane. First, fetal cartilage progenitor cell pellets were cultured in vitro for 3 days, then cultured in vitro, subcutaneously, and in an IVB for 3 weeks. First, the components and liquidity of IVB fluid were evaluated, then the chondrogenesis and immunogenicity of the pellets were evaluated using gross observation, cell viability assays, histology, biochemical analysis, RT-PCR, and Western blots. Finally, cartilage repair and synovial inflammation were evaluated histologically. RESULTS: The fluid color and transparency of the IVB were similar to synovial fluid (SF) and the components were closer to SF than serum. The IVB system not only promoted the synthesis of cartilage matrix and maintained the cartilage phenotype, it also delayed calcification compared to the subcutaneously implanted pellets. CONCLUSION: The IVB adopted to study cell differentiation was effective in preventing host immune rejection.

Sizable Scaffold-Free Tissue-Engineered Articular Cartilage Construct for Cartilage Defect Repair.

This study introduces an implantable scaffold-free cartilage tissue construct (SF) that is composed of chondrocytes and their self-produced extracellular matrix (ECM). Chondrocytes were grown in vitro for up to 5 weeks and subjected to various assays at different time points (1, 7, 21, and 35 days). For in vivo implantation, full-thickness defects (n = 5) were manually created on the trochlear groove of the both knees of rabbits (16-week old) and 3 week-cultured SF construct was implanted as an allograft for a month. The left knee defects were implanted with 1, 7, and 21 days in vitro cultured scaffold-free engineered cartilages. (group 2, 3, and 4, respectively). The maturity of the engineered cartilages was evaluated by histological, chemical and mechanical assays. The repair of damaged cartilages was also evaluated by gross images and histological observations at 4, 8, and 12 weeks postsurgery. Although defect of groups 1, 2, and 3 were repaired with fibrocartilage tissues, group 4 (21 days) showed hyaline cartilage in the histological observation. In particular, mature matrix and columnar organization of chondrocytes and highly expressed type II collagen were observed only in 21 days in vitro cultured SF cartilage (group 4) at 12 weeks. As a conclusion, cartilage repair with maturation was recapitulated when implanted the 21 day in vitro cultured scaffold-free engineered cartilage. When implanting tissue-engineered cartilage, the maturity of the cartilage tissue along with the cultivation period can affect the cartilage repair.

Adipose-derived stem cell spheroid treated with low-level light irradiation accelerates spontaneous angiogenesis in mouse model of hindlimb ischemia.

BACKGROUND AIMS: We investigated whether low-level light irradiation (LLLI) before adipose-derived stromal cells (ASCs) spheroid transplantation improved hind-limb functional recovery by stimulation of angiogenesis. METHODS: The spheroid, composed of ASCs, was irradiated with low-level light and expressed angiogenic factors, including vascular endothelial growth factor and basic fibroblast growth factor. From immunochemical staining analysis, the spheroid of ASCs included CD31+, KDR+ and CD34+, whereas monolayer-cultured ASCs were negative for these markers. To evaluate the therapeutic effect of the ASC spheroid treated with LLLI in vivo, phosphate-buffered saline, monolayer ASCs, LLLI-monolayer ASCs, spheroid ASCs and LLLI-spheroid ASCs were transplanted into a hind-limb ischemia model. RESULTS: The LLLI-spheroid ASCs transplanted into the hind-limb ischemia differentiated into endothelial cells and remained differentiated. Transplantation of LLLI-spheroid ASCs into the hind-limb ischemia significantly elevated the density of vascular formations through angiogenic factors released by the ASCs and enhanced tissue regeneration at the lesion site. Consistent with these results, the transplantation of LLLI-spheroid ASCs significantly improved functional recovery compared with ASC or spheroid ASC transplantation and PBS treatment. CONCLUSIONS: These findings suggest that transplantation of ASC spheroid treated with LLLI may be an effective stem cell therapy for the treatment of hind-limb ischemia and peripheral vascular disease.

Human adipose-derived stem cell spheroid treated with photobiomodulation irradiation accelerates tissue regeneration in mouse model of skin flap ischemia.

Skin flap grafting is a form of transplantation widely used in plastic surgery. However, ischemia/reperfusion injury is the main factor which reduces the survival rate of flaps following grafting. We investigated whether photobiomodulation (PBM) precondition prior to human adipose-derived stromal cell (hASC) spheroid (PBM-spheroid) transplantation improved skin tissue functional recovery by the stimulation of angiogenesis and tissue regeneration in skin flap of mice. The LED had an emission wavelength peaked at 660 ± 20 nm (6 J/cm2, 10 mW/cm2). The expression of angiogenic growth factors in PBM-spheroid hASCs was much greater than that of not-PBM-treated spheroid or monolayer-cultured hASCs. From immunochemical staining analysis, the hASCs of PBM-spheroid were CD31+, KDR+, and CD34+, whereas monolayer-cultured hASCs were negative for these markers. To evaluate the therapeutic effect of hASC PBM-spheroid in vivo, PBS, monolayer-cultured hASCs, and not-PBM-spheroid were transplanted into a skin flap model. The animals were observed for 14 days. The PBM-spheroid hASCs transplanted into the skin flap ischemia differentiated into endothelial cells and remained differentiated. Transplantation of PBM-spheroid hASCs into the skin flap ischemia significantly elevated the density of vascular formations through angiogenic factors released by the skin flap ischemia and enhanced tissue regeneration at the lesion site. Consistent with these results, the transplantation of PBM-spheroid hASCs significantly improved functional recovery compared with PBS, monolayer-cultured hASCs, and not-PBM-spheroid treatment. These findings suggest that transplantation of PBM-spheroid hASCs may be an effective stem cell therapy for the treatment of skin flap ischemia.

Angiogenic Synergistic Effect of Adipose-Derived Stromal Cell Spheroids with Low-Level Light Therapy in a Model of Acute Skin Flap Ischemia.

Human adipose-derived mesenchymal stem cells (hASCs) are an attractive cell source for tissue engineering. However, one obstacle to this approach is that the transplanted hASC population can decline rapidly in the recipient tissue. The aim of this study was to investigate the effects of low-level light therapy (LLLT) on transplanted spheroid hASCs in skin flaps of mice. hASCs were cultured in monolayers or spheroids. LLLT, hASCs, spheroids and spheroids transplanted with LLLT were applied to the skin flaps. Healing of the skin flaps was assessed by gross evaluation and by hematoxylin and eosin staining and elastin van Gieson staining. Compared with the spheroid group, skin flap healing was enhanced in the spheroid + LLLT group, including the neovascularization and regeneration of skin appendages. The survival of hASCs was enhanced by decreased apoptosis of hASCs in the skin flaps of the spheroid + LLLT group. The secretion of growth factors was stimulated in the spheroid + LLLT group compared with the ASC and spheroid groups. These data suggest that LLLT was an effective biostimulator of spheroid hASCs in the skin flaps, enhancing the survival of hASCs and stimulating the secretion of growth factors.

Enhancement of Ischemic Wound Healing by Spheroid Grafting of Human Adipose-Derived Stem Cells Treated with Low-Level Light Irradiation.

We investigated whether low-level light irradiation prior to transplantation of adipose-derived stromal cell (ASC) spheroids in an animal skin wound model stimulated angiogenesis and tissue regeneration to improve functional recovery of skin tissue. The spheroid, composed of hASCs, was irradiated with low-level light and expressed angiogenic factors, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF), and hepatocyte growth factor (HGF). Immunochemical staining analysis revealed that the spheroid of the hASCs was CD31+, KDR+, and CD34+. On the other hand, monolayer-cultured hASCs were negative for these markers. PBS, human adipose tissue-derived stromal cells, and the ASC spheroid were transplanted into a wound bed in athymic mice to evaluate the therapeutic effects of the ASC spheroid in vivo. The ASC spheroid transplanted into the wound bed differentiated into endothelial cells and remained differentiated. The density of vascular formations increased as a result of the angiogenic factors released by the wound bed and enhanced tissue regeneration at the lesion site. These results indicate that the transplantation of the ASC spheroid significantly improved functional recovery relative to both ASC transplantation and PBS treatment. These findings suggest that transplantation of an ASC spheroid treated with low-level light may be an effective form of stem cell therapy for treatment of a wound bed.

Adipose-derived stromal cell cluster with light therapy enhance angiogenesis and skin wound healing in mice.

Human adipose-derived mesenchymal stem cells (hASCs) are attractive cell source for skin tissue engineering. The aim of this study was to investigate the effects of low-level light therapy (LLLT) on transplanted cluster hASC in a skin wound animal model. The hASCs were cultured in monolayer or clusters. The LLLT, hASCs, hASC clusters, and hASC clusters transplantation with LLLT (cluster + LLLT) were applied to the wound bed in athymic mice. Wound healing was assessed by gross evaluation and by hematoxylin and eosin staining, and elastin van gieson histochemistry. The survival, differentiation, and secretion of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF), and hepatocyte growth factor (HGF) of the cluster ASC were evaluated by immunohistochemistry and Western blotting. The cluster + LLLT group enhanced the wound healing, including neovascularization and regeneration of skin appendages, compared with the cluster group. The secretion of growth factors was stimulated in the cluster + LLLT group compared with the ASCs and cluster group. These data suggest that LLLT is an effective biostimulator of cluster hASCs in wound healing that enhances the survival of hASCs and stimulates the secretion of growth factors in the wound bed.

Prevention of skin flap necrosis by use of adipose-derived stromal cells with light-emitting diode phototherapy.

BACKGROUND AIMS: The aim of this study was to investigate the effects of low-level light therapy (LLLT) on transplanted human adipose-derived mesenchymal stromal cells (ASCs) in the skin flap of mice. METHODS: LLLT, ASC transplantation and ASC transplantation with LLLT (ASC + LLLT) were applied to the skin flap. Immunostaining and Western blot analysis were performed to evaluate cell survival and differentiation and secretion of vascular endothelial growth factor and basic fibroblast growth factor by the ASCs. Vascular regeneration was assessed by means of immunostaining in addition to hematoxylin and eosin staining. In the ASC + LLLT group, the survival of ASCs was increased as the result of the decreased apoptosis of ASCs. RESULTS: The secretion of growth factors was higher in this group as compared with ASCs alone. ASCs contributed to tissue regeneration through vascular cell differentiation and secretion of angiogenic growth factors. The ASC + LLLT group displayed improved treatment efficacy including neovascularization and tissue regeneration compared with ASCs alone. Transplanting ASCs to ischemic skin flaps improved therapeutic efficacy for ischemia treatment as the result of enhanced cell survival and paracrine effects. CONCLUSIONS: These data suggest that LLLT is an effective biostimulator of ASCs in vascular regeneration, which enhances the survival of ASCs and stimulates the secretion of growth factors in skin flaps.